The ability to isolate viral DNA and clone this isolated DNA into bacterial plasmids has greatly expanded the approaches available to make viral vaccines. The methods used to make the present invention involve modifying cloned viral DNA sequences by insertions, deletions and single or multiple base changes. The modified DNA is then reinserted into the viral genome to render the virus non-pathogenic. The resulting live virus may then be used in a vaccine to elicit an immune response in a host animal and to protect the animal against a disease. One group of animal viruses, the herpesviruses or Herpetoviridae, is an example of a class of viruses amenable to this approach. These viruses contain 100,000 to 200,000 base pairs of DNA as their genetic material. Importantly, several regions of the genome have been identified that are nonessential for the replication of virus in vitro in cell culture. Modifications in these regions of the DNA may lower the pathogenicity of the virus, i.e., attenuate the virus. For example, inactivation of the thymidine kinase gene renders human herpes simplex virus non-pathogenic (13), and pseudorabies virus of swine non-pathogenic (14).
Removal of part of the repeat region renders human herpes simplex virus non-pathogenic (16,17). A repeat region has been identified in Marek's disease virus that is associated with viral oncogenicity (18). A region in herpesvirus saimiri has similarly been correlated with oncogenicity (19). Removal of part of the repeat region pseudorabies virus non-pathogenic (U.S. Pat. No. 4,877,737, issued Oct. 31, 1989). A region in pseudorabies virus has been shown to be deleted in naturally-occurring vaccine strains (7,15) and it has been shown that these deletions are at least partly responsible for the lack of pathogenicity of these strains.
It is generally agreed that herpesviruses contain nonessential regions of DNA in various parts of the genome, and that modifications of these regions can attenuate the virus, leading to a non-pathogenic strain from which a vaccine may be derived. The degree of attenuation of the virus is important to the utility of the virus as a vaccine. Deletions which cause too much attenuation of the virus will result in a vaccine that fails to elicit an adequate immune response. Although several examples of attenuating deletions are known, the appropriate combination of deletions is not readily apparent.
The natural host of pseudorabies virus is swine, in which infection is commonly not apparent but may be characterized by fever, convulsions and paralysis. Pseudorabies virus also infects cattle, sheep, dogs, cats, foxes and mink, where infection usually results in death of the host. The predominant visible feature of pseudorabies viral infection is intense pruritus generally resulting in host mutilation of the involved area. Violent excitement, fits and paralysis, all symptoms of encephalomyelitis, precede death which usually occurs within a few days following onset of clinical signs.
Pseudorabies virus disease in swine is of serious concern to governmental bodies worldwide. In the United States, swine from infected herds cannot be sold except to slaughterhouses. The U.S. Department of Agriculture has enacted an eradication program to eliminate pseudorabies. Prior to the development of specific differential vaccines and companion diagnostic tests, any animal vaccinated for pseudorabies was treated as though it were infected and was subjected to the same regulatory constraints. With the advent of differential vaccines, regulations have been modified to allow interstate shipment of vaccinated, non-infected swine provided the differential vaccine/diagnostic test combination has been approved for use in the Cooperative State-Federal Pseudorabies Eradication Program (Federal Register, Vol. 55, No. 90, pp. 19245-19253 (May 9, 1990)).
The construction of differential vaccines has focused on the deletion of one of the glycoproteins of PRV. Theoretically, the glycoprotein chosen to be the diagnostic marker should have the following characteristics: (1) the glycoprotein and its gene should be non-essential for the production of infectious virus; (2) the glycoprotein should elicit a strong and persistent response in the animal; (3) the glycoprotein should not make a contribution to the protective immune response. Three glycoproteins gD, gH, and gB(II) (25) fail the first criteria. Each has a counterpart in herpes simplex virus (HSV) which has proven to be essential (20). g63 is a minor glycoprotein and would not be expected to elicit a major serological response (8). gIII has been shown to make a significant contribution to protective immunity as a target of neutralizing antibodies (10) and as a target of cell-mediated immunity (21). gI is also the target of neutralizing antibodies (22) and might be expected to play a role in protective immunity. Only gpX meets all three of the theoretical criteria (25).
Currently, there are five USDA-licensed, modified live, differential PRV vaccines available. They are PRV/Marker (SyntroVet Incorporated), Tolvid (Upjohn), PRV vaccine (Boehringer Ingelheim), PRVac (SmithKline Beecham Animal Health) and OmniMark (Fermenta Animal Health). The companion diagnostic test for each of these vaccines is directed against one of three PRV glycoproteins (gpX, gI or gIII). In each case, the gene coding for the diagnostic glycoprotein has either been altered by genetic engineering or has been shown to be deleted in a naturally occurring virus. The diagnostic marker deleted from PRV/Marker and Tolvid is gpX, which was altered by genetic engineering techniques. PRV vaccine and PRVac contain viruses that have a spontaneous deletion of gI from a naturally occurring virus. gIII is deleted from OmniMark, as a result of genetic engineering.
As the pseudorabies eradication program progresses, it would be of great value to have a confirmatory diagnostic test. Due to differing antigenicity of each diagnostic antigen and to the nature of the immune response of individual animals, the level of antibody to the diagnostic antigen can vary widely. A vaccine which incorporates a second diagnostic marker which could be used in a confirmatory test would be of great value. Two virus strains have been described which have incorporated the deletion of two glycoproteins for the purposes of serologic differentiation. One of these, described by Kit et al. (U.S. Pat. No. 4,711,850), has a genetically engineered deletion of gIII and a naturally occurring deletion of gI. As discussed above, both of these glycoproteins are targets of neutralizing antibody and gIII is also the target of cell-mediated immunity. The deletion of both of these important antigens would be expected to compromise the efficacy of a vaccine. The second virus, described by Post et al. (25) has been genetically engineered to incorporate deletions in both the gpX and gI genes. The authors concluded that this virus was significantly compromised in efficacy relative to a virus in which only the gpX was deleted. In summary, the current state of the art indicates that a virus deleted in both gpX and gI would not be effective as a vaccine.
A vaccine superior to the currently available products would have the following characteristics: (1) not produce clinical signs in 3-4 day old piglets; (2) give 95% protection in pigs of all ages; (3) permit serological differentiation from wild-type infected animals; and (4) permit a confirmatory diagnostic test. This invention provides such a superior vaccine, unexpectedly, by deleting specific regions of both gpX and gI.